PROJECT SUMMARY: Our laboratory studies the essential process of mRNA decay in the model Gram- positive bacterium, Bacillus subtilis. Rapid turnover of mRNA fragments is required to replenish ribonucleotide pools and to avoid non-productive translation on mRNA fragments. Relative to what is known about bacterial transcription and translation, much less is known about mRNA decay. All bacterial species contain a 3'-to-5' exoribonuclease, which degrades mRNA fragments that are generated by a decay-initiating endonuclease cleavage. Much evidence suggests that the key 3'-to-5' exonuclease in B. subtilis is polynucleotide phosphorylase (PNPase), encoded by the pnpA gene. RNA-Seq data show that 5'-proximal RNA fragments for hundreds of genes accumulate in a pnpA deletion strain. The current proposal aims to understand the specificity of mRNA turnover by PNPase and what compensates for PNPase in a pnpA deletion strain. Structural studies suggest that RNA is threaded from its 3' end into a central channel of PNPase, which can only bind single-stranded RNA. Thus, efficient decay of mRNA that contains secondary structure may depend on RNA helicase activity. Preliminary data show that the major B. subtilis RNA helicase, CshA (encoded by the cshA gene) is required for rapid decay of some mRNA fragments. We propose to use new RNA-Seq protocols in B. subtilis ?pnpA and ?cshA strains to discover the nature of mRNA sequences that determine susceptibility to PNPase and dependence on CshA. The results of RNA-Seq experiments will guide genetic and biochemical experiments that probe the requirements for efficient PNPase-mediated decay. Available tools will be used to explore the relationship of PNPase-mediated decay to RNase Y (the major decay-initiating endonuclease), to ribosome flow, and to the ribosome rescue system. In addition, a unique screening method for determining susceptibility to decay is proposed. The screen promises to give insight into the nature of RNA sequences that are efficiently degraded by PNPase, and when there is a need for helicase activity. Despite considerable accumulation of RNA fragments, a pnpA knockout strain grows well, indicating that some 3' exonuclease compensates for the loss of PNPase. The other three known 3' exoribonucleases of B. subtilis are likely not significantly involved in mRNA turnover. In preliminary experiments, we detect at least one 3' exonuclease activity in an extract of a strain that is missing all four of the known 3' exonucleases. The identity of this activity will be pursued using a biochemical approach, and its function in mRNA turnover will be studied. Discovery of an additional processive 3' exonuclease, which is not predicted from genome sequence, will impact greatly on our understanding of bacterial RNA processing and decay. RELEVANCE: Degradation of messenger RNA is an essential function of bacteria. A thorough understanding of the mechanism of mRNA decay will enable design of antimicrobial agents that disrupt this process and thereby interfere with bacterial cell growth.